Although studies in cell lines have identified the main components of the mammalian apoptotic pathway, the differences in the regulation of apoptosis among various primary cells remain largely unexplored. For example, biochemical and molecular studies have identified the caspase proteases as central executioners of apoptosis and the release of mitochondrial holocytochrome c (heme-attached cytochrome c) as a critical trigger that activates caspases during apoptosis. However, despite the central importance of cytochrome c- mediated caspase activation in mammalian apoptosis, our understanding of how this pathway is regulated in primary cells is very limited. Our recent results point to a potentially fundamental difference in the regulation of caspase activation in primary mitotic versus postmitotic cells. We find that whereas holocytochrome c alone is necessary and sufficient to activate caspases and induce apoptosis in mitotic cells, it is necessary but not sufficient to induce apoptosis in neurons, cardiomyocytes, and myotubes. This difference is striking and is indicative of novel, differential mechanisms of apoptosis regulation in mitotic versus postmitotic cells. Unfortunately however, our understanding of the molecular basis for this difference is limited because the only current method for activating apoptosis at the point of cytochrome c is via single cell microinjection of holocytochrome c. Thus, these experiments are restricted to only those cells that can be microinjected and they are not compatible with large scale biochemical analysis. To overcome these limitations and to develop novel probes for studying apoptosis regulation, we will adapt a cell-free caspase activation assay for high throughput compatibility to screen for small-molecule compounds that trigger apoptosis after the point of mitochondrial cytochrome c release in cells. The development of these chemical probes is significant in two important aspects: a) They are essential tools for discovering the molecular mechanism of the differential regulation of apoptosis in mitotic and postmitotic cells, and b) These probes are predicted to have therapeutic value as reagents that induce apoptosis only in selective cells.